It is well known that a prolonged and high alcohol intake can lead to serious alcohol-related diseases and alcoholism. A low to moderate intake is, however, nowadays commonly accepted as beneficial due to the associated reduced risk of coronary heart disease. In order to assess a persons drinking behavior, e.g. for classifying this drinking behavior as healthy or unhealthy, relatively slowly eliminating direct bio-markers of ethanol is highly useful. One such bio-marker that has gained considerable interest over the last years is phosphatidylethanol (PEth).
PEth is an abnormal metabolite, formed by ethanolysis of precursor phospholipids with a glycerol backbone under catalytic influence of phospholipase D in the presence of ethanol. PEth exists in the form of several similar homologues, each with a unique set of long chain carboxylic acid residues as substituents. These homologues are commonly named in the form “PEth AA:B/CC:D”, wherein AA indicates the number of carbons in the carboxylic acid substituent at the first position of the glycerol backbone and B indicates the number of double bonds encompassed by that carbon chain; CC indicates the number of carbons in the carboxylic acid substituent at the second position of the glycerol backbone and D indicates the number of double-bonds encompassed by that carbon chain. This nomenclature, however, does not indicate the position, nor the stereochemistry, of any present double-bonds. For example, the regio- and stereo-isomers (Z)-1-(palmitoyloxy)-3-([ethoxy(hydroxy)phosphoryl]oxy)prop-2-yl octadec-12-enoate and (E)-1-(palmitoyloxy)-3-([ethoxy(hydroxy)phosphoryl]oxy)prop-2-yl octadec-9-enoate are both named “PEth 16:0/18:1”.
Clinically used analytical methods for the detection of PEth in blood-samples include HPLC-methods with evaporative light scattering (ELS) detection. A sum of different PEth-homologues are typically detected and used as a basis for quantification of PEth-levels.
Other analytical methods include methods which employ MS-detection of one or several selected homologues of PEth (see for example A. Tolonen et al, Analytical Biochemistry 2005, 341, 83-88). Sample throughput is usually greater in these analytical methods in comparison to methods which employ ELS-detection or similar techniques. Another advantage of methods with MS-detection is a lower detection limit of PEth in a sample, which allows for determination of a lower amount of previously consumed ethanol in a test subject. The MS-detector is typically tuned to discriminate between compounds based on their molecular weight in order to detect one or a few of the relatively more commonly occurring PEth-homologues.
Commonly occurring PEth-homologues in human blood after ethanol intake include 16:0/18:1 and 16:0/18:2.
WO2009054784A1 describes a method for assessing previous ethanol intake by measurement of PEth-homologues comprising mainly 16:0-, 18:0-, 18:1-, 18:2- or 20:4-carboxylic acid substituents, by employment of capillary electrophoresis and e.g. UV-detection.
Disadvantages of above mentioned methods for the determination of previous ethanol intake by the quantification of one or several PEth-homologues, include a high risk for erroneous conclusions regarding e.g. the amount of previously consumed ethanol due to the natural variability in PEth-homologue composition.
In addition, all of the above mentioned methods rely on simultaneous detection of several different PEth-homologues, or on the detection of one or a few selected PEth-homologues under the assumption that this selection gives a correct view of previous ethanol intake.
Hence, improved methods for the determination of previous ethanol intake are desired.